Comparative genomics analysis coupled to functional assays uncovered evolutionarily conserved signaling pathways and provided important insights towards the understanding of insect immunity. However, extrapolating this approach to non-insect arthropods, such as the tick Ixodes scapularis, sometimes constitutes a problem because of the lack of distinguishable protein homologues and incorrect annotation of genes due to low sequence coverage or incompleteness of a particular genome. For instance, we observed that the immune deficiency (IMD) signaling pathway of the tick I. scapularis is critical for defense against the rickettsial agent Anaplasma phagocytophilum despite the absence of the adaptor molecule imd and the gene fadd on its genome. Moreover, we learned that the E3 ubiquitin ligase x-linked inhibitor of apoptosis protein (XIAP) is critical for activating the IMD pathway against A. phagocytophilum infection of I. scapularis ticks. Collectively, these findings led to ou central hypothesis stating that XIAP regulates the non-canonical tick IMD pathway in response to the rickettsial agent A. phagocytophilum. Accordingly, in Aim #1 of this proposal, we will characterize the functional interaction between XIAP and the E2-conjugating enzyme Bendless. We surmise a direct interaction between these two proteins based on structural docking and mass spectrometry analysis. In Aim #2, we will identify XIAP substrates during pathogen colonization of ticks using stable isotope labeling with amino acids in cell culture (SILAC) and diglycine remnant affinity profiling combined with tandem mass spectrometry. In Aim #3, we will validate the IMD network during A. phagocytophilum infection of I. scapularis ticks in vivo. We will ascertain the immune response following pathogen entry into the tick and evaluate the effect of the IMD signaling pathway during pathogen infection. The outcome of this research will be two-fold: (1) it will provide fundamental knowledge related to tick-pathogen interactions; and (2) it will uncover a novel immune pathway in arthropods with broad implications for the vector biology community.